The goal of this project is to characterize the shape, molecular weight distribution and elemental composition of specific individual macromolecules and macromolecular assemblies. Such assemblies are critical to many cell functions, and their behavior in vitro reflects their function and regulation in intact cells. This project depends on a unique instrument - a low~temperature, high~resolution, field~emission scanning transmission electron microscope (STEM)-for molecular weight mapping and chemical analysis by parallel electron energy loss spectroscopy (EELS) of directly frozen thin films and ultrathin cryosections of directly frozen tissues. Dark~field molecular weight mapping of native neurofilaments (NFs) from squid axoplasm indicated that the mass per unit length of squid NFs was 38=B15 kDa/nm, similar to values reported for other NF assemblies. Second~difference EELS spectra revealed that there are 30 P atoms within the 10~nm region of the NF, corresponding to complete phosphorylation of available KSP sites on the heavy subunit of these filaments. These results indicate that EELS is capable of detecting physiologically relevant differences in phosphorylation states at a useful spatial resolution of 10~20 nm. We have applied a new method~based on analyzing the valence electron region of a low~dose EELS map of frozen~hydrated sections to determine the distribution of water within mouse liver and within cerebellar cortex that had undergone trauma~induced cellular swelling. This method in combination with high~dose EELS spectrum imaging is expected to reveal trauma~related changes in water and calcium distributions at the subcellular level.